ONCOLOGY REPORTS 32: 2354-2358, 2014
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Circulating miR-199a-3p as a novel serum biomarker for colorectal cancer Ryoji Nonaka1, Junichi Nishimura1, Yoshinori Kagawa1, Hideki Osawa1, Junichi Hasegawa2, Kohei Murata3, Shu Okamura3, Hirofumi Ota4, Mamoru Uemura1, Taishi Hata1, Ichiro Takemasa1, Tsunekazu Mizushima1, Daisuke Okuzaki5, Hirofumi Yamamoto1, Yuichiro Doki1 and Masaki Mori1 1
Department of Surgery, Gastroenterological Surgery, Graduate School of Medicine, Osaka University, Osaka; Department of Surgery, Osaka Rosai Hospital, Osaka; 3Department of Surgery, Suita Municipal Hospital, Osaka; 4 Department of Surgery, Saiseikai Senri Hospital, Osaka; 5Special Research Facilities, DNA-chip Development Center for Infectious Diseases, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
2
Received March 31, 2014; Accepted July 7, 2014 DOI: 10.3892/or.2014.3515 Abstract. Serum microRNAs (miRNAs) have been shown to have potential for cancer diagnosis. The main objective of the present study was to identify a novel serum miRNA biomarker from patients with colorectal cancer (CRC). Microarray analysis of miRNA expression was performed using paired pre-operative and post-operative serum from 10 CRC patients. Expression of two miRNAs (let-7a and miR-199a-3p) was significantly decreased in the post-operative serum when compared to levels in the pre-operative serum (P=0.015 and 0.029, respectively). Quantitative real-time polymerase chain reaction (qRT-PCR) confirmed the decrease in the miRNAs in an extended number (n=30) of paired serum samples. Next, we examined the serum let-7a level in 32 non-cancer patients and 84 CRC patients but we found no significant difference (P= 0.120). In contrast, miR199a-3p expression was significantly higher in the CRC patients than that in the non-cancer patients (P=0.016). Furthermore, clinical and pathological survey indicated that high expression of miR-199a-3p was significantly associated with deep wall invasion. Our data suggest that circulating miR-199a-3p could be a novel serum biomarker for CRC.
Correspondence to: Dr Hirofumi Yamamoto, Department of Surgery, Gastroenterological Surgery, Graduate School of Medicine, Osaka University, 2-2 Yamadaoka, Suita City, Osaka 565-0871, Japan E-mail: [email protected]
Abbreviations: CRC, colorectal cancer; miR/miRNA, microRNA; qRT-PCR, quantitative reverse transcription-polymerase chain reaction
Key words: miR-199a-3p, circulating miR, colorectal cancer
Introduction Colorectal cancer (CRC) is one of the leading causes of cancerrelated mortality worldwide. In spite of screening examination, CRC incidence and the associated mortality have increase rapidly in the past several decades (1,2). The prognosis of CRC is associated with stage, and CRC can be completely cured if detected early; the 5-year survival rate is 93.2% for stage I, and 8.1% for stage IV (3). Thus, early detection of CRC is crucial to reduce mortality, and CRC-related deaths can be prevented through early detection and early treatment. Several CRC screening tests including fecal occult blood testing (FOBT) and colonoscopy have been available for years (4). However, these methods are associated with issues such as low adherence rates, high cost or low sensitivity. An ideal screening method should have a high sensitivity and specificity for early stage CRC; it should also be safe and accepted by patients (5-7). MicroRNAs (miRNAs) are 22-nucleotide non-coding RNA molecules that regulate a variety of cellular processes including cell differentiation, cell cycle progression and apoptosis. miRNAs have been demonstrated to play an important role in the multistep processes of carcinogenesis either by oncogenic or tumor-suppressive function. The study of miRNAs has been extended to many types of tumors, including CRC (8-10). These studies have revealed that miRNAs may be potential diagnostic or prognostic tools for human cancers. Tumor-associated RNAs have been described in the serum/plasma of cancer patients for more than a decade. More recently, several studies have also demonstrated that circulating miRNAs exist in serum/plasma (11,12). Accordingly, several subsequent studies have proven that miRNAs can serve as potential biomarkers for various diseases including cancer. It has been revealed that miR-92 and miR-21 are significantly elevated in the plasma of CRC patients and can be potential non-invasive molecular markers for CRC detection (13-16). In the present study, we aimed to identify a novel circulating miRNA in CRC patients and to evaluate its feasibility as a noninvasive diagnostic test for efficient detection of CRC.
NONAKA et al: Circulating miR-199a-3p in colorectal cancer
2355
Materials and methods Patients and samples. Informed consent was obtained from CRC patients and non-cancer patients for the use of their blood samples. From April 2011 to June 2013, venous blood samples were collected from the CRC patients (n=114) and non-cancer patients who suffered inguinal hernia or gall bladder stone (n=32). In 30 of the CRC patients, blood samples were obtained before and on the 7th day after surgery. No cancer patients received chemotherapy or radiotherapy before blood sampling. The blood samples were obtained from Osaka University, Osaka Rosai Hospital, Suita Municipal Hospital and Saiseikai Senri Hospital. Whole blood was collected, centrifuged at 1,000 rpm and 4˚C for 15 min. The supernatant fluids were centrifuged at 15,000 rpm and 4˚C for 10 min. The supernatant fluids were stored at -80˚C until RNA extraction. This study was conducted under the supervision of the Ethics Board of Osaka University Hospital. RNA extraction. Small RNA was enriched from all serum samples using the mirVana Paris RNA isolation kit (Ambion, Austin, TX, USA), following the manufacturer's instructions. Briefly, 400 µl of serum was thawed on ice and centrifuged at 15,000 rpm for 15 min to remove cell debris. Next, 300 µl of the supernatant was lysed with an equal volume of 2X denaturing solution. For normalization of sample-to-sample variation during the RNA isolation procedures, 20 fmol of synthetic C. elegans miRNA cel-miR-39 was added to each denatured sample. Small RNAs were then enriched and purified following the manufacturer's protocol. The concentration of all RNA samples were quantified by NanoDrop ND-1000 (Nanodrop, Wilmington, DE, USA). miRNA microarray analysis. miRNA microarray experiments were carried out using Agilent human miRNA microarray catalogued in the Sanger database ver. 12.0 (design ID 021827). Approximately 10 ng of aliquots of total RNA with cel-miR-39 was used for making the miRNA probes according to the Agilent protocol (ver. 2.3). Microarrays were performed for paired pre-operative and post-operative serum from 10 CRC patients. Briefly, total RNA was dephosphorylated with calf intestine alkaline phosphatase, denatured with dimethyl sulfoxide, and labeled with pCp-Cy3 using T4 RNA ligase using an miRNA labeling reagent and hybridization kit. Probes were hybridized at 55˚C for 20 h with rotation. Then the slides were washed by Gene Expression Wash Buffer 1 at room temperature for 5 min and by Gene Expression Wash Buffer 2 at 37˚C for 5 min. After hybridization and washing, the slides were scanned using an Agilent scanner (G2505C). Images were extracted using Agilent Feature Extraction software (ver. 10.7.3.1) and Agilent GeneSpring GX software (ver. 10.0.2). Differences in miRNA expressions between the 10 pairs was determined when the fold-change of cel-miR-39 normalized expression values was >2.0 and the P-value was
2354
Circulating miR-199a-3p as a novel serum biomarker for colorectal cancer Ryoji Nonaka1, Junichi Nishimura1, Yoshinori Kagawa1, Hideki Osawa1, Junichi Hasegawa2, Kohei Murata3, Shu Okamura3, Hirofumi Ota4, Mamoru Uemura1, Taishi Hata1, Ichiro Takemasa1, Tsunekazu Mizushima1, Daisuke Okuzaki5, Hirofumi Yamamoto1, Yuichiro Doki1 and Masaki Mori1 1
Department of Surgery, Gastroenterological Surgery, Graduate School of Medicine, Osaka University, Osaka; Department of Surgery, Osaka Rosai Hospital, Osaka; 3Department of Surgery, Suita Municipal Hospital, Osaka; 4 Department of Surgery, Saiseikai Senri Hospital, Osaka; 5Special Research Facilities, DNA-chip Development Center for Infectious Diseases, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan
2
Received March 31, 2014; Accepted July 7, 2014 DOI: 10.3892/or.2014.3515 Abstract. Serum microRNAs (miRNAs) have been shown to have potential for cancer diagnosis. The main objective of the present study was to identify a novel serum miRNA biomarker from patients with colorectal cancer (CRC). Microarray analysis of miRNA expression was performed using paired pre-operative and post-operative serum from 10 CRC patients. Expression of two miRNAs (let-7a and miR-199a-3p) was significantly decreased in the post-operative serum when compared to levels in the pre-operative serum (P=0.015 and 0.029, respectively). Quantitative real-time polymerase chain reaction (qRT-PCR) confirmed the decrease in the miRNAs in an extended number (n=30) of paired serum samples. Next, we examined the serum let-7a level in 32 non-cancer patients and 84 CRC patients but we found no significant difference (P= 0.120). In contrast, miR199a-3p expression was significantly higher in the CRC patients than that in the non-cancer patients (P=0.016). Furthermore, clinical and pathological survey indicated that high expression of miR-199a-3p was significantly associated with deep wall invasion. Our data suggest that circulating miR-199a-3p could be a novel serum biomarker for CRC.
Correspondence to: Dr Hirofumi Yamamoto, Department of Surgery, Gastroenterological Surgery, Graduate School of Medicine, Osaka University, 2-2 Yamadaoka, Suita City, Osaka 565-0871, Japan E-mail: [email protected]
Abbreviations: CRC, colorectal cancer; miR/miRNA, microRNA; qRT-PCR, quantitative reverse transcription-polymerase chain reaction
Key words: miR-199a-3p, circulating miR, colorectal cancer
Introduction Colorectal cancer (CRC) is one of the leading causes of cancerrelated mortality worldwide. In spite of screening examination, CRC incidence and the associated mortality have increase rapidly in the past several decades (1,2). The prognosis of CRC is associated with stage, and CRC can be completely cured if detected early; the 5-year survival rate is 93.2% for stage I, and 8.1% for stage IV (3). Thus, early detection of CRC is crucial to reduce mortality, and CRC-related deaths can be prevented through early detection and early treatment. Several CRC screening tests including fecal occult blood testing (FOBT) and colonoscopy have been available for years (4). However, these methods are associated with issues such as low adherence rates, high cost or low sensitivity. An ideal screening method should have a high sensitivity and specificity for early stage CRC; it should also be safe and accepted by patients (5-7). MicroRNAs (miRNAs) are 22-nucleotide non-coding RNA molecules that regulate a variety of cellular processes including cell differentiation, cell cycle progression and apoptosis. miRNAs have been demonstrated to play an important role in the multistep processes of carcinogenesis either by oncogenic or tumor-suppressive function. The study of miRNAs has been extended to many types of tumors, including CRC (8-10). These studies have revealed that miRNAs may be potential diagnostic or prognostic tools for human cancers. Tumor-associated RNAs have been described in the serum/plasma of cancer patients for more than a decade. More recently, several studies have also demonstrated that circulating miRNAs exist in serum/plasma (11,12). Accordingly, several subsequent studies have proven that miRNAs can serve as potential biomarkers for various diseases including cancer. It has been revealed that miR-92 and miR-21 are significantly elevated in the plasma of CRC patients and can be potential non-invasive molecular markers for CRC detection (13-16). In the present study, we aimed to identify a novel circulating miRNA in CRC patients and to evaluate its feasibility as a noninvasive diagnostic test for efficient detection of CRC.
NONAKA et al: Circulating miR-199a-3p in colorectal cancer
2355
Materials and methods Patients and samples. Informed consent was obtained from CRC patients and non-cancer patients for the use of their blood samples. From April 2011 to June 2013, venous blood samples were collected from the CRC patients (n=114) and non-cancer patients who suffered inguinal hernia or gall bladder stone (n=32). In 30 of the CRC patients, blood samples were obtained before and on the 7th day after surgery. No cancer patients received chemotherapy or radiotherapy before blood sampling. The blood samples were obtained from Osaka University, Osaka Rosai Hospital, Suita Municipal Hospital and Saiseikai Senri Hospital. Whole blood was collected, centrifuged at 1,000 rpm and 4˚C for 15 min. The supernatant fluids were centrifuged at 15,000 rpm and 4˚C for 10 min. The supernatant fluids were stored at -80˚C until RNA extraction. This study was conducted under the supervision of the Ethics Board of Osaka University Hospital. RNA extraction. Small RNA was enriched from all serum samples using the mirVana Paris RNA isolation kit (Ambion, Austin, TX, USA), following the manufacturer's instructions. Briefly, 400 µl of serum was thawed on ice and centrifuged at 15,000 rpm for 15 min to remove cell debris. Next, 300 µl of the supernatant was lysed with an equal volume of 2X denaturing solution. For normalization of sample-to-sample variation during the RNA isolation procedures, 20 fmol of synthetic C. elegans miRNA cel-miR-39 was added to each denatured sample. Small RNAs were then enriched and purified following the manufacturer's protocol. The concentration of all RNA samples were quantified by NanoDrop ND-1000 (Nanodrop, Wilmington, DE, USA). miRNA microarray analysis. miRNA microarray experiments were carried out using Agilent human miRNA microarray catalogued in the Sanger database ver. 12.0 (design ID 021827). Approximately 10 ng of aliquots of total RNA with cel-miR-39 was used for making the miRNA probes according to the Agilent protocol (ver. 2.3). Microarrays were performed for paired pre-operative and post-operative serum from 10 CRC patients. Briefly, total RNA was dephosphorylated with calf intestine alkaline phosphatase, denatured with dimethyl sulfoxide, and labeled with pCp-Cy3 using T4 RNA ligase using an miRNA labeling reagent and hybridization kit. Probes were hybridized at 55˚C for 20 h with rotation. Then the slides were washed by Gene Expression Wash Buffer 1 at room temperature for 5 min and by Gene Expression Wash Buffer 2 at 37˚C for 5 min. After hybridization and washing, the slides were scanned using an Agilent scanner (G2505C). Images were extracted using Agilent Feature Extraction software (ver. 10.7.3.1) and Agilent GeneSpring GX software (ver. 10.0.2). Differences in miRNA expressions between the 10 pairs was determined when the fold-change of cel-miR-39 normalized expression values was >2.0 and the P-value was
